Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, India.
J Virol. 2024 Aug 20;98(8):e0085824. doi: 10.1128/jvi.00858-24. Epub 2024 Jul 30.
Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3'-NCR of JEV genomic RNA containing the GQ sequence could bind NCL . The replication complex binding to the 3'-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication.IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3'-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.
日本脑炎病毒（JEV）是一种节肢动物传播的正链黄病毒，可导致人类病毒性脑炎，病死率高。JEV 非结构蛋白 5（NS5）具有 RNA 依赖性 RNA 聚合酶活性，与病毒和宿主蛋白相互作用构成复制复合物。我们已鉴定出多功能蛋白核仁素（NCL）是几种 NS5 相互作用的宿主蛋白之一。我们证明了 JEV NS5 与病毒感染的 HeLa 细胞中的 NCL 相互作用和共定位。siRNA 介导的 NCL 敲低表明其对病毒复制是必需的。重要的是，在过表达外源性 NCL 的细胞中，JEV 的滴度更高，表明其具有促进病毒的作用。我们证明 NS5 与 NCL 的 RRM 和 GAR 结构域相互作用。我们表明，含有 G-四链体（GQ）结构的 NCL 结合适体 AS1411 和 GQ 配体 BRACO-19 可显著抑制 JEV 复制。在 HeLa 细胞中，外源性 NCL 的过表达可克服 AS1411 和 BRACO-19 的抗病毒作用。我们证明源自 JEV 基因组 RNA 3'-NCR 的含有 GQ 序列的合成 RNA 可与 NCL 结合。复制复合物与 3'-NCR 的结合是病毒 RNA 合成所必需的。很可能是复制复合物中的 NCL 使基因组 RNA 中的 GQ 结构不稳定，从而促进复制复合物的移动，导致病毒复制效率提高。重要性日本脑炎病毒（JEV）在东南亚和西太平洋地区的大部分地区流行，可引起脑炎流行，病死率高。虽然有组织培养衍生的 JEV 疫苗，但尚无抗病毒疗法。JEV NS5 蛋白具有 RNA 依赖性 RNA 聚合酶活性。与几种宿主和病毒蛋白一起，它构成了病毒复制所必需的复制复合物。了解 NS5 与宿主蛋白的相互作用有助于设计新型抗病毒药物。我们鉴定出核仁素（NCL）是 JEV NS5 的关键宿主蛋白相互作用蛋白，在病毒复制中具有促进病毒的作用。与 NS5 相互作用的 NCL 结合到 JEV RNA 的 3'-NCR 中的 G-四链体（GQ）结构序列。这可能使复制复合物沿基因组 RNA 的运动更加顺畅，从而促进病毒复制。这项研究首次报道了宿主蛋白 NCL 如何通过 GQ 结合帮助 JEV 复制。
